Nitrate (NO3−) supplementation has been reported to enhance intermittent exercise performance; however, its impact on oxygen (O2) cost during intermittent running exercise is unclear. The aim of this study was to assess if acute NO3− supplementation would elicit performance benefits in recreationally active individuals during the Yo–Yo intermittent recovery level 1 (Yo-Yo IR1) test, with its potential benefit on O2 consumption (VO2), in a double-blind, randomized, crossover study, 12 recreational males consumed NO3−-rich (NIT; ~12.8 mmol), and NO3−-depleted (PLA; 0.04 mmol) concentrated beetroot juice 3 h before completing the Yo-Yo IR1 test. VO2 was measured at 160, 280 and 440 m (sub-maximal) and when the test was terminated (peak). Performance in the Yo–Yo IR1 was greater with NIT (990 ± 442.25 m) compared to PLA (870 ± 357.4 m, p = 0.007). The VO2 was not significantly different at 160 m (1.92 ± 0.99 vs. 2.1 ± 0.88 L·min−1), 280 m (2.62 ± 0.94 vs. 2.83 ± 0.94 L·min−1), 440 m (3.26 ± 1.04 vs. 3.46 ± 0.98 L·min−1) and peak (4.71 ± 1.01 vs. 4.92 ± 1.17 L·min−1) between NIT and PLA trials (all p > 0.05). The present study has indicated that acute supplementation of NO3− enhanced intermittent running performance but had no effect on VO2 during the Yo–Yo IR1 test in recreational young adults.